Blood was collected by cardiac puncture at the time of animal sacrifice, and a nonselective COX inhibitor, indomethacin, was immediately added to the blood samples together with the anticoagulant sodium citrate. The plasma was separated by spinning the samples at 5000 × g for 4 min. CysLTs and PGE2 plasma samples were measured using a competitive enzyme immunoassay obtained from Enzo Life Sciences (Solna, Sweden). All measurements were performed according to the manufacturer’s instructions.
Plasma cytokines were analyzed using a multiplex sandwich immunoassay format and the electro-chemiluminescence MSD ultrasensitive proinflammatory multiplex kit (Meso-Scale Discovery, Gaithersburg, MD). The MSD multispot array was run according to the manufacturer’s protocol. Briefly, 96-well plates pre-coated with capture antibodies for TNFα, IL-1β, IL-2, IL-4, IL-6, IL-10 and CXCL1/KC/GRO, INFγ were incubated with plasma samples for 2 h. Subsequently, detection antibodies were added, and the plate was incubated for another 2 h. After washing, the plate was read using an MS2400 imager (MSD).
Plasma cytokines were analyzed using a multiplex sandwich immunoassay format and the electro-chemiluminescence MSD ultrasensitive proinflammatory multiplex kit (Meso-Scale Discovery, Gaithersburg, MD). The MSD multispot array was run according to the manufacturer’s protocol. Briefly, 96-well plates pre-coated with capture antibodies for TNFα, IL-1β, IL-2, IL-4, IL-6, IL-10 and CXCL1/KC/GRO, INFγ were incubated with plasma samples for 2 h. Subsequently, detection antibodies were added, and the plate was incubated for another 2 h. After washing, the plate was read using an MS2400 imager (MSD).